Distribution of most ancient CNEs across phyla. Cladogram showing presence (green), absence (red), or mixed presence-absence (orange) of the deeply conserved RPLP1 and RPLP2 CNEs. Numbers in brackets show number of species analyzed per group for the RPLP1/RPLP2 CNEs respectively. Groups are outlined by color: blue (Protostomia), orange (Deuterostomia), green (Cnidaria, Ctenophora, and Placozoa), pink (Porifera) 

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... 6 Evolution of RPLP1 CNE over at least 670 million years. a Alignment of RPLP1 CNE in all organisms where detected. Sequence logo diagrams of each conserved motif are shown below the alignment. Motif 1a is protostome-specific whereas motif 1b appears to be the ancestral and deuterostome form. Motifs 2 and 3 are variably spaced and are present in all phyla examined. Species name color scheme matches that of Fig. 5. b Diagram showing the position and spacing of each motif in each organism in relation to the translation start site. Genus/species abbreviations are defined in Additional file 2: Table S10  ...

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... 7 Evolution of RPLP2 CNE over at least 700 million years. a Alignment of RPLP2 CNE in all organisms where detected. The three distinct sequence motifs are shown aligned below the main alignment. Species name color scheme matches that of Figure 5. b Diagram showing the position and spacing of each motif in each organism in relation to the translation start site. Genus/species abbreviations are defined in Additional file 2: Table S10  ...

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... deeply conserved sequences (> = 350 Myr) also appear to be associated with a specific class of genes. 14 of these CNEs were found to lie completely within transcribed regions (see Methods), and all 20 were found to overlap transcribed regions by at least a third of the length of the CNE. This enrichment is significant ( p < 6.5e-04, hypergeometric test) when compared to the full set of 322, of which only ~70 % overlap transcribed regions by this amount. Remarkably for such a small set of genes, a GO term overrepresentation test turned up 39 significant terms. The 17 genes associated with these 20 CNEs are enriched for genes active in processes such as post-transcriptional regulation of gene expression (q < 6.1e-4), regulation of translation (q < 4.8e-3), and translational elongation (q < 1.2e-2). This list of overrepresented GO terms is, unlike that obtained from the full set of 322 CNEs, devoid of terms relating to transcriptional regulation, matching the shift towards putative translational regulatory CNEs. Among the CNEs that we identified were previously- studied regulatory elements, as well as many unidentified novel putative regulatory elements. As the majority of CNEs overlap 5 ’ UTRs, we calculated the likelihood of there being a conserved secondary structure in each CNE. This analysis revealed several conserved secondary structures, including an example of the well-characterized iron response element (IRE) in the 5 ’ UTR of the Ferritin gene (Additional file 1: Figure S4), a conserved hairpin loop bound by iron response proteins (IRPs) to help maintain iron homeostasis. We also identified novel conserved RNA structures, including a conserved, strong ( − 52.60 kcal/mol) hairpin loop found in the 5 ’ UTR of the Paramyosin gene (Additional file 1: Figure S5) identified in all four Hymenoptera species, and a hairpin loop with perfect stem complementarity but variable apical sequence conserved in the 5 ’ UTR of the Not1 gene (Fig. 4). These three hairpins differ in their fundamental characteristics. IREs are characterized by a highly conserved apical sequence (CAGUGY; clearly demonstrated in the three hymenopteran species) with a more variable stem sequence [3]. In contrast, the 4- nucleotide apical sequence (consensus HVHN) of Not1 appears to be highly variable, whereas the stem sequence is almost perfectly conserved. More sequences are necessary to be able to reliably characterize the Paramyosin hairpin, although there does appear to be at least one variable nucleotide in the hairpin apex. The positions of the hairpins also appear to be of functional importance; all three hairpins are conserved in their position relative to the translation start site, particularly the Not1 hairpin (Fig. 4a). The Not1 hairpin loop has a stem sequence of 12 bp, and the CNE containing it is found directly adjacent to the translation start site. The CNE contains two conserved stem sequences with near-perfect complementarity, a weakly conserved apical sequence, and a highly conserved, upstream ATG-containing motif directly adjacent to the translation start site. In N. vitripennis , this CNE is present in the 5 ’ UTR of all four known transcripts. As the position of this CNE is so strongly conserved, we scanned the first 100 bp of every orthologous transcript in all Ensembl Metazoa species for presence of either the conserved hairpin or for the conserved sequence adjacent to the translation start site. The results of this search (see Methods) indicated that in all cases where the hairpin loop is present the conserved sequence adjacent to the translation start site is present too, but not vice-versa (i.e. the sequence adjacent to the translation start site may exist on its own). The presence of the sequence in the Antarctic krill Euphasia superba (Hunt and Rosato, unpublished data) and in a centipede ( Strigamia maritima ) shows that this CNE was an early arthropod adaptation. We identified conserved putative regulatory sequences in six separate genes of the insect-specific Osiris gene cluster (Additional file 1: Figure S6). Our analysis indicates that these regions are Hymenoptera-specific, and are generally conserved in position relative to their associated gene. Since the conserved regions are associated with a specific class of genes with core functions, the fact that conserved promoter regions were identified near to six genes in the same cluster is perhaps indicative of an important developmental or regulatory role for this as-yet uncharacterized gene cluster. Two conserved sequences were identified in the 5 ’ UTRs of the two ribosomal stalk heterodimer genes, RPLP1 and RPLP2 . Given that parts of these sequences were found to be perfectly conserved over several nucleotides, we looked for presence of the same sequences in more distant phyla. A motif elicitation analysis (see Methods) revealed three separate sequence motifs in the RPLP1 CNE, and three in the RPLP2 CNE. These motifs are present in many different phyla (Fig. 5), including both deuterostomes and protostomes, making these the third and fourth known examples of bilaterian conserved regulatory elements (Bicores) [13]. These two conserved sequences were both early innovations in Animalia. The RPLP1 CNE was found in the genomes of the placozoan Trichoplax adhaerens and the cnidarian Nematostella vectensis (starlet sea anemone). N. vectensis also contains the RPLP2 CNE, as do the ctenophore Mnemiopsis leidyi (warty comb jelly) and the poriferan Amphimedon queen- slandica (a demosponge). Both CNEs were present in the majority of species that we analyzed ( RPLP1 : 33/38 species analyzed, RPLP2 : 23/38). Previously, the most ancient CNEs identified were found conserved between Deuterostomia and Cnidaria [11], thus dating back over 670 million years [12]. The CNE on RPLP2 that we report here appears to have originated even earlier, being found in the Porifera. This CNE is thus likely over 700 million years old [8]. The CNE on RPLP1 may also be older than 670 million years, depending on how the deep splits in the phylogeny of animals are eventually resolved [18]. The conserved regions paint an interesting evolutionary story. Firstly, in the RPLP1 CNE (Fig. 6), there are three distinct conserved sequences. The first conserved sequence appears to have two distinct forms; one found in protostomes (motif 1a) and another in deuterostomes (motif 1b), which appears to be the ancestral form as it is found in Cnidaria and Placozoa. The sec- ond and third motifs are found well conserved across both deuterostomes and protostomes, and are variably spaced; for example all mammalian species analyzed share a similar insertion between these two motifs. The relative position of the CNE is found conserved across all phyla (Fig. 6b), remaining within 150 bp of the translation start site. The RPLP2 CNE (Fig. 7) also appears to be described best as three distinct motifs. Motif 1 is exceptionally well conserved, with no variation at all across 10 bp. Motif 2 comprises a conserved region, generally followed by a short stretch of adenine nucleotides. Motif 3 is short and does not appear to be present in either D. melanogaster or Mnemiopsis leidyi . These observations make clear that these CNEs are functionally complex, being com- prised of several discrete elements punctuated by less evolutionarily constrained sequence. This is in contrast to other kinds of conservation such as ultraconserved regions, where long stretches of nucleotides (>200 bp) are found perfectly conserved between human, rat, and mouse,[19] which can be in some cases deleted without a clear critical loss of function [20]. As a whole, the complexity, shared associated gene function, and age of these CNEs marks them as interesting targets for future study. In this paper, we used a high stringency statistical approach to identify and characterize 322 ancient noncoding elements (Additional file 2: Table S4) which have remained conserved over large evolutionary distances. The bulk of the conserved sequences that we identified are specific to Hymenoptera, but nevertheless have been conserved in place for at least 180 million years of insect evolution (which occurs at a faster pace than vertebrate evolution [14]). A small proportion of the CNEs (20) that we identified were at least 350 million years old, with three stretching back further still. Two CNEs are found conserved in a range of the most basal animal clades across a wide variety of both vertebrates and in- vertebrates and are likely over 670 [12] and 700 million years old [8], likely the oldest CNEs described to date. These two ancient CNEs are located in the 5 ’ UTRs of two genes that are known to interact with one another; RPLP1 and RPLP2 . The two protein products of these ubi- quitously expressed genes, P1 and P2, form a heterodimer; two copies of which bind to the 60s acidic ribosomal protein P0 (coded by the gene RPLP0 ) to form the ribosomal stalk. The ribosomal stalk is involved in translational fine tuning and is crucial for the correct folding of many proteins [21]. The depth and breadth of conservation of these sequences is indicative of a fundamental regulatory role. Indeed, the 5 ’ UTR of RPLP2 has already been shown to have a regulatory role in Drosophila [22], being sufficient to confer full translational control unto RPLP2 as a non- translated gene in the early embryo, but not previously known to be conserved among animals. The fact that this CNE has been previously studied and identified as a regulatory element helps to validate the idea that other CNEs that we have identified are also functional regulatory elements. In Drosophila , the CNE we identified essentially spans the entirety of the RPLP2 5 ’ UTR, whereas in other organisms it is only a constituent part. In this study, we have characterized the motifs likely to be important for the function of this regulatory element and examined their evolution over time. Most of the conserved regions ...